No abstract

Biotransformation of natural products has great potential for producing new drugs and could provide in vitro models of mammalian metabolism. Microbial transformation of the cytotoxic steroid cinobufagin was investigated. Cinobufagin could be specifically hydroxylated at the 12␤-position by the fungus Alternaria alternata. Six products from a scaled-up fermentation were obtained by silica gel column chromatography and reversed-phase liquid chromatography and were identified as 12␤-hydroxyl cinobufagin, 12␤-hydroxyl desacetylcinobufagin, 3-oxo-12␤-hydroxyl cinobufagin, 3-oxo-12␤-hydroxyl desacetylcinobufagin, 12-oxo-cinobufagin, and 3-oxo-12␣-hydroxyl cinobufagin. The last five products are new compounds. 12␤-Hydroxylation of cinobufagin by A. alternata is a fast catalytic reaction and was complete within 8 h of growth with the substrate. This reaction was followed by dehydrogenation of the 3-hydroxyl group and then deacetylation at C-16. Hydroxylation at C-12␤ also was the first step in the metabolism of cinobufagin by a variety of fungal strains. In vitro cytotoxicity assays suggest that 12␤-hydroxyl cinobufagin and 3-oxo-12␣-hydroxyl cinobufagin exhibit somewhat decreased but still significant cytotoxic activities. The 12␤-hydroxylated bufadienolides produced by microbial transformation are difficult to obtain by chemical synthesis.Bufadienolides are steroids with a characteristic ␣-pyrone ring at the C-17 position; they have cardiotonic, blood pressure-stimulating, antiviral, and local anesthetic activities (20). More than 300 bufadienolides have been isolated from natural sources including plants and animals (15,23). These compounds have been reported to have significant antitumor activities (9,19,31,33). Cinobufagin (compound I) is a bufadienolide with a 14␤,15␤-epoxy ring, originally isolated as a major component of the traditional Chinese drug, Chan'Su (also called toad venom or toad poison), which is prepared from the skin secretions of giant toads (10). Cinobufagin can induce apoptosis and elevate intracellular Ca 2ϩ levels (13,14). Against cancer cells it has a 50% inhibitory concentration (IC 50 ) of approximately 10 Ϫ8 mol/liter (30). Unfortunately, it is poorly soluble in water and is toxic to humans; therefore, analogs with improved pharmaceutical properties, such as those obtained from plant cell suspension cultures (27,29), are needed before this compound can be utilized effectively in a clinical setting.Microbial transformation is an important tool for structural modification of organic compounds, especially natural products with complicated structures (17,22). It can be used to synthesize chemical structures that are difficult to obtain by other means (24) and as a model of mammalian metabolism due to the similarity between mammalian and microbial cytochrome P 450 enzyme systems (1,2,4,5,12). The metabolism of cinobufagin in rat liver microsomes produced at least six metabolites (32), resulting primarily from deacetylation at C-16 and epimerization of 3-OH via a 3-keto intermediate. The exact s...

Thirteen compounds were isolated from the anomalous fruits of Gleditsia sinensis on the basis of bioassay-guided fractionation. These saponins together with six analogues or related compounds were tested for their cytotoxicities against six tumor cell lines by the MTT method. The induction of apoptosis in HL-60 cells by these compounds was determined through flow cytometric analysis. Some structure-activity relationships in cytotoxicity and induction of apoptosis were identified. The evaluation of the cytotoxicity and the ability to induce apoptosis revealed that some important structural features are required for activity. A valuable model which enables prediction of their activities was established and may be employed for the drug design of new Gleditsia saponin analogues.

Previous studies have shown that pseudolaric acid B (PB) would cause apoptosis in human tumor cell lines. However, the mechanisms of PB induced apoptosis are still unclear. In the present study, the mechanisms of PB induced apoptosis in the human hepatocellular carcinoma Bel-7402 cell line were investigated by measuring cell viability, rate of apoptosis, cell cycle, detecting DNA fragmentation, and measuring caspase-3 activation. The results indicated that PB inhibited Bel-7402 cell viability and induced cell death by causing DNA fragmentation, up regulating the early and late apoptotic rates, activating caspase-3 protein, and detaining the cell cycle in the G2/M phases. Additionally, PB-induced apoptosis was a dose- and time-dependent manner. These observations suggest that PB-induced apoptosis occurs through a caspase-dependent pathway and detains the cell cycle in the G2/M phase.

). † These authors contributed equally to this work. SUMMARYChitinases are a class of ubiquitous proteins that are widely distributed in plants. Defense is the major natural role for chitinases, primarily against fungal pathogens. Little is known regarding their non-defensive roles in seeds. In this study, a new class III chitinase from pomegranate seeds (pomegranate seed chitinase, PSC) was isolated and purified to homogeneity. The native state of PSC is a monomer with a molecular weight of approximately 30 kDa. This chitinase naturally binds calcium ions with high capacity and low affinity, suggesting that PSC is a calcium storage protein. Consistent with this idea, its amino acid sequence (inferred from cDNA) is rich in acidic amino acid residues, especially Asp, similar to reported calcium storage proteins. The presence of calcium considerably improves the stability of the protein but has little effect on its enzymatic activity. Transmission electron microscopy analyses indicate that, similar to phytoferritin, this enzyme is widely distributed in the stroma of amyloplasts of the embryonic cells, suggesting that amyloplasts in seeds could serve as an alternative plastid for calcium storage. Indeed, the transmission electron microscopy results showed that, within the embryonic cells, calcium ions are mainly distributed in the stroma of the amyloplasts, consistent with a role for PSC in calcium storage. Thus, the plant appears to have evolved a new plastid for calcium storage in seeds. During seed germination, the content of this enzyme decreases with time, suggesting that it is involved in the germination process.